Benoit Lefebvre

Summary: Fungal plant diseases are a major cause of crop loss worldwide. Understanding the processes driving these diseases is therefore crucial to anticipate and control epidemics while improving crop yield production. Moreover, the new European « Green Deal » aims to reduce the use of pesticides, while, few alternatives are currently available on the market. There is therefore an urgent need to better track and anticipate fungal crop epidemics in the current context of fungicide reduction. In light of this, our study aims to understand Fusarium spill overs from non-agricultural habitats in order to better characterise potential non-host Fusarium reservoirs that may be implicated in the emergence of Fusarium diseases. To address this question, we isolated Fusarium lineages from natural populations of Brassica rapa . We characterised their phylogenomic affiliation and found that they display high diversity, with a population structure related to the locality of isolation. By inoculation assays, we identified Fusarium strains from non-agricultural habitats that were highly aggressive on wheat. In addition, we observed a strong effect of wheat variety on disease development. We monitored the Fusarium colonisation through a microscopy approach in both wheat and Brassica plants. We observed that while the fungus is mostly on root surface in Brassica plants, it spreads inter-cellularly in wheat roots with vascular colonisation in the wheat shoots. Finally, we showed that most aggressive Fusarium strains were isolated from Brittany, a French region where wheat is vastly cultivated.

The below-ground microbiota plays a pivotal role in enhancing plant resilience to environmental stresses. Investigating microbial diversity within native agricultural systems is essential for understanding the potential of microbiota in sustaining crop health and productivity. Agricultural practices, including the selection of plant genotypes, are key determinants in shaping plant-microbe interactions. However, the role of plant genetic diversity in structuring below-ground microbiota has been only partially explored. Furthermore, many studies examining crop-microbiota interactions have been conducted under controlled conditions. In this study, we performed a two-year in situ survey of root and rhizosphere microbiota in 35 wheat ( Triticum aestivum) genotypes grown in open fields under different organic agricultural practices, including unfertilized conditions, organic fertilization, and intercropping with faba bean. We characterized both bacterial and fungal communities and found that plant genotype, along with organic fertilization, were significant factors influencing the diversity and composition of root and rhizosphere microbiota. In addition, we showed that landraces varieties harbor a more diversified fungal microbiota when compared to modern varieties. Our study underscores the importance of incorporating wheat genetic diversity into agricultural strategies to enhance microbiota diversity.

Chitooligosaccharides, such as chitin, are essential components of fungal cell walls and have thus naturally been selected as microbe-associated molecular patterns detected by plants to initiate defense mechanisms. These molecules are typically recognized by the lysin motif receptor-like kinases (LysM RLKs) at the plasma membrane. While chitin perception is well elucidated in Arabidopsis thaliana and other plant species, the recognition mechanisms of its deacetylated form, chitosan, remain poorly investigated despite its use as a biocontrol strategy to protect crops against pathogens. Here, we investigated the role of the two grapevine orthologs of AtLYK4, which participate in the tripartite complex for chitin perception in A. thaliana. Using a dual approach consisting of the functional complementation of the atlyk4/5 double mutant and CRISPR-Cas9 genome editing in Vitis vinifera, we showed that VvLYK4-2 is involved in both chitosan- and chitin-induced immune responses, encompassing MAPK phosphorylation and defense gene expression. Furthermore, grapevine in vitro plantlets lacking VvLYK4-2 exhibited a significantly reduced response to chitosan while retaining a low-intensity response to chitin, potentially due to the presence of VvLYK5-1. Finally, VvLYK4-2 produced in a heterologous system showed binding to chitosan oligomers and, to a lesser extent, to chitin oligomers. These findings indicate that this pattern recognition receptor plays a crucial role in the perception of chitosan oligomers and has thus potential for selective breeding purposes. This discovery may also help to better understand the partial lack of efficacy of chitosan-based plant defense stimulants used in viticulture.

Background and aims: Arbuscular mycorrhizal fungi (AMF) play an essential role in plant nutrition in both natural and agro-ecosystems. However, how soil nutrient availability simultaneously regulates AMF diversity and contribution to plant nutrition, requires more attention. We hypothesised that the interaction between the availability of key soil macronutrients phosphorus (P) and nitrogen (N) regulates AMF contribution to wheat nutrition and that nutrient availability will simultaneously influence AMF community composition. Methods: We tested this using a unique long-term P fertilisation trial, sampling wheat roots across two growing seasons. Expression of wheat mycorrhizal nutrient transporters was quantified by RT-qPCR and AMF communities were characterised by ITS2 metabarcoding. Complementary experiments under controlled conditions examined how the interaction between P and N regulates arbuscular mycorrhizal function in plant nutrition. Results: Field-grown wheat showed campaign-specific effects of P fertilisation on AMF colonisation and nutrient transporter expression, which coincided with shifts in plant N status. Controlled experiments confirmed that colonisation depends on the limitation of either P or N, but that the regulation of peri-arbuscular phosphate, ammonium and nitrate transporters depended on the limiting nutrient. AMF communities also responded to soil P availability, with the genus Funneliformis consistently dominating under high P conditions. Conclusions: Our findings demonstrate that P and N availability jointly shape root AMF communities and regulate their nutritive function in wheat. The combination of community profiling and mycorrhizal molecular markers provides a valuable approach for understanding the AMF contribution to plant nutrition across agroecosystems, and therefore can be used for optimising agroecological practices.

Understanding the contributions of arbuscular mycorrhizal fungi (AMF) to plant nutrition is essential for sustainable agriculture. We hypothesised that soil phosphorus (P) availability modulates the diversity and functionality of wheat root-associated AMF community, particularly the mycorrhizal nutrition. Wheat plants were sampled over two campaigns (2019 and 2022) in a long-term P fertilisation trial. The expression of the wheat transporters involved in the mycorrhizal nutrition was assessed by RT-qPCR, and AMF community composition by metabarcoding. Complementary experiments under controlled conditions were performed to further study the interaction between nitrogen (N) and P availability on AM. Different effects of P on AMF colonisation and transporter expression were observed in field-grown wheat depending on the campaigns, which differed in wheat N status. Controlled experiments confirmed that AMF colonisation depends on the limitation of either P or N, but that regulation of peri-arbuscular phosphate, ammonium and nitrate transporters depends on the nature of the limiting soil nutrients. Additionally, AMF communities varied according to the soil P availability, with the Funneliformis genus becoming more dominant under high P conditions for both years. Together, our findings show that N and P availability jointly shape root AMF communities and AM functioning. Combining community profiling and molecular markers of colonisation and nutrition offers a framework to better understand AMF contributions to plant nutrition across agroecosystems.

Many plants interact symbiotically with arbuscular mycorrhizal fungi to enhance inorganic phosphorus uptake, and legumes also develop a nodule symbiosis with rhizobia for nitrogen acquisition. The establishment and functioning of both symbioses rely on a common plant signaling pathway activated by structurally related Myc and Nod factors. Recently, a SPARK receptor-like kinase (RLK)/receptor-like cytoplasmic kinase (RLCK) complex was shown to be essential for arbuscular mycorrhiza formation in both monocot and dicot plants. Here, we show that in Aeschynomene legumes, the RLCK component of this receptor complex has undergone a gene duplication event and mediates a unique nodule symbiosis that is independent of rhizobial Nod factors. In Aeschynomene evenia, AeRLCK2 is crucial for nodule initiation but not for arbuscular mycorrhiza symbiosis. Additionally, AeRLCK2 physically interacts with and is phosphorylated by the cysteine-rich RLK, AeCRK, which is also required for nodulation. This finding uncovers an important molecular mechanism that controls the establishment of nodulation and is associated with Nod-independent symbiosis.

Lipo-chitooligosaccharides (LCO) and short-chain chitooligosaccharides (CO) are produced by arbuscular mycorrhizal fungi (AMF) and activate the plant symbiosis signalling pathway, which is essential for mycorrhiza formation. High-affinity LCO receptors belonging to the LysM receptor-like kinase (LysM-RLK) phylogenetic group LYR-IA play a role in AM establishment, but it is unclear which proteins are the plant high-affinity short-chain CO receptors. Here we studied members of the uncharacterized LYR-IB group, and found that they show high affinity for LCO, short- and long-chain CO, and play a complementary role with the LYR-IA receptors for AM establishment. While LYR-IB knock out mutants had a reduced AMF colonization in several species, constitutive/ectopic expression in wheat increased AMF colonization. LYR-IB function is conserved in all tested angiosperms, but in most japonica rice a deletion creates a frameshift in the gene, explaining differences in AM phenotypes between rice and other monocot single LYR-IA mutants. In conclusion, we identified a class of LysM-RLK receptors in angiosperms with unique biochemical properties and a role in both LCO and CO perception for AM establishment.

Arbuscular mycorrhiza (AM) with soilborne Glomeromycota fungi was pivotal in the conquest of land by plants almost half a billion years ago. In flowering plants, it is hypothesized that AM is initiated by the perception of AM fungi-derived chito- and lipochito-oligosaccharides (COs/LCOs) in the host via Lysin Motif Receptor-Like Kinases (LysM-RLKs). However, it remains uncertain whether plant perception of these molecules is a prerequisite for AM establishment and for its origin. Here, we made use of the reduced LysM-RLK complement present in the liverwort Marchantia paleacea to assess the conservation of the role played by this class of receptors during AM and in CO/LCO perception. Our reverse genetic approach demonstrates the critical function of a single LysM-RLK, MpaLYKa, in AM formation, thereby supporting an ancestral function for this receptor in symbiosis. Binding studies, cytosolic calcium variation recordings and genome-wide transcriptomics indicate that another LysM-RLK of M. paleacea , MpaLYR, is also required for triggering a response to COs and tested LCOs, despite being dispensable for AM formation. Collectively, our results demonstrate that the perception of symbionts by LysM-RLK is an ancestral feature in land plants, and suggest the existence of yet-uncharacterized AM fungi signals.

Botrytis cinerea is recognized as one of the most damaging fungal pathogens affecting grapevine ( Vitis vinifera ), directly impacting both grape yield and wine quality. Identifying new genes involved in the interaction between V. vinifera and B. cinerea appears to be a promising strategy for enhancing grapevine resistance in future breeding programs. During pathogen infection, plasma membrane-localized pattern recognition receptors (PRRs) are responsible for detecting conserved microbe-associated molecular patterns (MAMPs). Among PRRs, members of the LysM receptor-like kinase family are well known to mediate the recognition of fungal MAMPs and triggering plant immune signaling pathways. Interestingly, a novel member of this receptor family, named VvLYK6, was identified in grapevine as the most highly upregulated during B. cinerea infection. To investigate the role of VvLYK6 in plant immunity, we conducted overexpression studies in Arabidopsis thaliana and grapevine cell suspensions. Overexpression of VvLYK6 led to a reduction in chitin-induced MAPK activation, decreased expression of defense-related genes, reduced callose deposition, and increased plant susceptibility to fungal pathogens in A. thaliana . Based on these findings, we conclude that VvLYK6 acts as a negatively regulator of chitin-triggered immune responses, suggesting its potential role as a susceptibility gene during fungal infections.

Summary: In addition to environmental factors, plant genetics play a key role in shaping the root microbiota; however, the extent of this genetic control remains underexplored. Using a collection of 181 wheat lines derived from a genetically diverse population, we investigated the influence of wheat genotypes on the composition of the root endophytic mycobiota and explored the genetic determinants driving these relationships. We first characterized the mycobiota associated with the roots of field-grown lines from the Evolutionary Prebreeding pOpulation (EPO) using Internal Transcribed Spacer 2 (ITS2) barcoding. Fungal diversity was then correlated with wheat genetics by quantitative methods, including heritability analysis and Genome Wide Association Studies (GWAS), to identify novel genetic determinants influencing the mycobiota composition. Fungal species richness showed a positive correlation across most fungal clades, except between Mortierellomycotina and Glomeromycotina . Some specific fungal clades, such as Olpidiomycota or Chytridiomycota , underscored their potential as root endophytes. Additionally, we observed higher heritability in fungal clades ( i.e. at the phylum or subphylum rank) that exhibit a homogenous trophic mode, such as the biotrophic Arbuscular Mycorrhizal Fungi (AMF). This study identifies 11 QTLs associated with mycobiota composition at the clade level. By shedding light on the genetic control of fungal diversity and uncovering key fungal associations, this work enhances our understanding of plant-microbiota interactions and highlights the potential for breeding strategies to optimize these relationships.